A number of pathogenic leptospires are known to be of economic importance because they cause disease in different animal species, for example, in dogs, cattle, pigs, sheep and horses. Notably, it is a common cause of abortion, stillbirth, and neonatal mortality in swine, of renal and hepatic disease in dogs. To prevent such disease, vaccines consisting of killed bacteria have been prepared from cultures of leptospires.
Leptospires are bacteria, including various serovars, and of these canicola, icterohaemorrhagiae, pomona, grippotyphosa, bratislava, australis, ballum, aulumnalis and hardjo are most frequently responsible for leptospirosis of animals and humans.
Leptospires are most often cultivated in protein-containing media. These media contain serum from various species, notably from rabbit, bovine, equine . . . . To prepare such vaccines, it would be desirable to have a high density of the leptospires in the culture medium and/or to achieve this high density by concentration procedure. After administration of a vaccine comprising such serum components, adverse reactions may occur. The risk of adverse systemic reactions is increased due to the concentration of the heterologous serum proteins, in particular serum albumin. The adverse systemic reactions include anaphylaxis, hypersensitivity and atypical reactions such as vomiting and diarrhea.
Various chemically defined protein-free media for the growth of leptospires have been proposed. In order to obtain the desired rapid and abundant growth of organisms necessary for the efficient production of vaccines, it has been necessary to supplement such media with a source of fatty acids, notably Tween®, metabolisable by leptospires. But the source of fatty acid may also be toxic for leptospires, so to reduce this disadvantage serum from animal origin or serum albumin was added to the culture media.
Fatty acids are the major carbon and energy source for leptospires (Bey R. F. and Johnson R. C., Infection and Immunity, 1978, 19(2), 562-569). In addition, long-chain fatty acids are essential nutrients because the leptospires are unable to synthesize these acids de novo. Although they can decrease the length of the carbon chain of fatty acids and they can desaturate fatty acids, the leptospires can not increase the length of these compounds. Because free fatty acids are toxic to the leptospires, serum or serum albumin is incorporated into culture media to bind the fatty acids in an available but nontoxic form. Media containing bovine serum albumin (BSA) and Tween® have most commonly been accepted among the manufacturers to produce Leptospires vaccines. Ellinghausen and McCullough (American Journal of Veterinary Research, 1965, 26(1), 39-44) have shown that Leptospires may be grown on a serum-free medium, provided a complex of albumin and lipid is supplied. These workers further (Ellinghausen and McCullough, American Journal of Veterinary Research, 1965, 26(1), 45-51) showed that Tween® 80 could serve as a lipid source for Leptospira pomona but that excess lipid (more than 0.1% of Tween® 80) caused a lysis of the leptospires and a falloff in their growth.
Vaccine production requires the use of large volumes of culture media, and the most expensive component is animal serum or serum albumin. The serum proteins are also the cause of adverse systemic vaccine reactions.
To reduce the systemic reactions, one can purify vaccines to remove components thereof which cause the systemic reactions. Animal vaccine preparations can typically be purified by conventional methods such as filtration, diafiltration or centrifugation to remove the supernatant. Other methods of purification that yield highly purified antigens are seldom employed because they are cost prohibitive in the preparation of animal vaccines. At any rate, these purification methods were not effective for removing albumin from vaccines or precursors thereof.
To circumvent the disadvantages of adverse systemic reactions and of the use of animal serum or serum albumin, a number of chemically defined media for the cultivation of leptospires have been proposed in the prior art.
Anion-exchange resins also binds fatty acids, and they can be used as a substitute for albumin for the cultivation of leptospires. The major disadvantage of using the resins in this manner is that they must be separated from the leptospires when they are harvested (Bey R. F. and Johnson R. C., Infection and Immunity, 1978, 19(2), 562-569).
In these attempts, Tween® (ethoxylated fatty acid ester of sorbitan, polysorbate) was usually employed as a fatty acid source in the metabolism of leptospires since the microorganism utilizes long-chain fatty acids. However, commercial Tween® is usually contaminated with unesterified fatty acids, polyethylene glycols and other by-products, and these materials impede the growth of leptospires. Hence removal of the contaminants from commercial Tween® by such methods as ion exchange resin treatment, charcoal-treatment and polyvinylpyrrolidone-treatment, was attempted.
Staneck et al. (Staneck et al., Infect. Immun., 1973, 7(6), 886-897) reported that albumin was not necessary for cultivation of L. canicola and L. Pomona if the Tween® 80 (polyoxyethylene sorbitan monooleate) was passed through an anion-exchange column to reduce its free-fatty acid content. However the attempts of Bey & Johnson to detoxify the Tween® by passage through an anion-exchange column were unsuccessful (Bey R. F. and Johnson R. C., Infection and Immunity, 1978, 19(2), 562-569).
Bey & Johnson reported the growth of leptospires in a synthetic medium containing charcoal treated Tween® (Bey R. F. and Johnson R. C., Infection and Immunity, 1978, 19(2), 562-569).
Polyvinylpyrrolidone (PVP)-treated Tween® was used to prepare a protein-free medium for cultivation of leptospires (Schönberg A., Zbl. Bakt. Hyg., 1983, 254, 540-544). PVP is a synthetic colloid showing a behaviour similar to that of serum protein. However some serovars of leptospires, notably L. grippotyphosa, did not grow in this medium.
Kojima et al. (Kojima et al., Microbiol. Immunol., 1984, 28(8), 949-954) reported the growth of leptospires in a synthetic medium containing detoxified Tween®. The detoxification was done by extraction of unesterified fatty acids with n-hexane.
The removal of the Tween® contaminant or the detoxification of Tween® was not easy. Notably due to the great variation of composition of the commercial Tween®, from supplier to another one and from lot to lot, these methods could be incomplete. More than that, these methods were time consuming and expensive.
Some experiments were attempt to grow leptospires directly in a protein-free medium and with untreated Tween®. Stalheim (Stalheim O. H. V., J. Bacteriol., 1966, 92(4), 946-951) wanted to grow leptospires in a medium without protein, notably without albumin and without any detoxifying agent, but with untreated Tween® as fatty acid source. Face to culture problem, Stalheim proposed to adapt these leptospires to survive and consume untreated Tween® by successive passages on culture media containing an increased concentration of untreated Tween® at each passage. After several subcultures, as long as 7 to 27 days per subculture, Leptospira pomona tolerated higher concentrations of Tween® 80 (0.06%). But a concentration of 0.1% of Tween® 80 was extremely lytic for Leptospires. With 0.06% of Tween® 80, the final biomass of Leptospires was only 2.0 10e8 bacteria/ml, which is inferior to the biomass obtained with classical culture method using treated Tween®, notably charcoal-treated Tween® 80, e.g. Leptospira canicola with 5 10e8 bacteria/ml or Leptospira icterohaemorrhagiae with 8.4 10e8 bacteria/ml (Bey and Johnson, supra).
So there is still a need for a better culture process for leptospires.
One objective of the invention is to provide an adaptation process of Leptospires to grow in a protein-free culture medium. A second objective of the present invention is to provide a new process for the large scale culture of Leptospires in a protein-free culture medium.